Summary
Many proteins and peptides are present at relatively low concentrations in complex biological samples such as body fluids or cellular extracts. In order to be able to inject a sufficient amount on the miniaturized analytical systems used in proteomics (e.g. nanoLC-MS), it is therefore necessary to enrich the target proteins and to eliminate interfering substances including proteins that are deemed not to be of interest.Affinity enrichment is based on structural features of a given group of proteins or peptides. Well-known examples are the enrichment of phosphorylated peptides based on their interaction with immobilized transition metal ions or the enrichment of glycoproteins on immobilized lectin columns. In this project we will focus on two post-translational modifications that are related to tumor development and inflammation, respectively:

a)    Enrichment of glycoproteins using lectin affinity chromatography: In our previous work we have shown that the profile of glycoproteins in serum of cervical cancer patients changes in comparison to healthy controls (unpublished data). We anticipate that these changes are related to tumor development, since it is known that glycosylation patterns change during tumorigenesis. We will use this disease target as an example to develop a novel, fully integrated glycoprotein profiling system based on affinity solid-phase extraction (SPE) followed by on-line trypsin digestion and chipLC-MS(/MS) analysis. The core of this system will be based on a prototype that we have developed for the activity-based profiling of metalloproteases in a collaboration with Hermen Overkleeft (Leiden).
b)    Chemical labeling of nitrotyrosine residues: A post-translational modification that is rather rare and has not been studied in great detail, is the formation of nitrotyrosine as a consequence of •NO-dependent oxidative stress during an inflammatory reaction. While most presently available approaches rely on monoclonal antibodies against nitrotyrosine, we have developed a chemical labeling strategy that relies on the tagging of nitrotyrosine-derived aminotyrosine residues (unpublished data). This strategy is based on a sequence of “one-pot” reactions, which run with very high yields. Based on our work on smoke-induced oxidative stress we will develop and apply this approach to proteomes from cells in culture that have been exposed to gas-phase cigarette smoke, cigarette smoke extract and air as control as a first proof of principle. We will subsequently extend our work to body fluids from patients exposed to oxidative stress due to a smoking episode.